Linear or cyclic aminophosphonates as pH markers in phosphorus 31 NMR spectroscopy

ABSTRACT

Linear or cyclic aminophosphonates are disclosed which are useful as pH markers. A method of using the linear or cyclic aminophosphonates in phosphorus-31 NMR spectroscopy is also disclosed.

The invention relates to novel linear or cyclic aminophosphonates and totheir use as pH markers in phosphorus-31 NMR spectroscopy, Moregenerally, the A invention relates to the use of aminophosphonatederivatives as pH markers in NMR spectroscopy. Phosphorus-31 NMRspectroscopy has been found to be an effective means for measuringextracellular and intracellular pH in vivo.

The advantage of this method is that it does not at all disrupt themedium on which the measurement is taken, which is an essentialcondition for an in vivo measurement.

A compound can be used as a pH marker when the numerical value of thechemical shift of the resonance peak obtained by ³¹P NMR varies as afunction of the pH of the medium into which the compound has beenintroduced. The difficulty consists in developing the ideal, non-toxiccompound which will be able to function as a pH marker in a broad pHrange, with good sensitivity. An additional requirement is that themeasurement should be little, if at all, affected by the otherconstituents of the physiological medium and that it should only reactto a pH variation, even a very small one.

A certain number of markers are commonly proposed in the art. The onemost commonly used is inorganic phosphate Pi, which has the advantage ofbeing an endogenous compound present in all cells. However, this markerhas two major drawbacks which may prevent the production of precisemeasurements (R. J. Gillies et al. (1986) Proc. Soc. Magn. Reson. Med.5, 153-154):

the Pi content is generally relatively low in the cell and varies withthe metabolic state of the cell;

the lack of sensitivity of this compound does not make it possible todistinguish between extra-cellular and intracellular pH.

2-Deoxyglucose 6-phosphate and methyl phosphonate have also been tested(M. DeFronzo et al., (1987) J. Biol. Chem. 262, 11032-11037). Theresults of these studies show that methyl phosphonate is a much moresensitive marker than 2-deoxyglucose 6-phosphate. Furthermore, althoughthe latter compound is not metabolized, it is found to be toxic to thecell. On the other hand, despite its low toxicity, methyl phosphonatehas the major drawback of total permeability with respect to cellmembranes in the case of the tumour cell line studied.

Phenyl phosphonate is another extracellular pH marker (cf. CirculationResearch, vol. 60, No. 4, 1987, 472-477). The drawback of this compoundis that the ³¹P chemical shift is influenced by the presence of specificions in the measuring medium. The American Physiological Society, 1994,C195-C203 moreover reports the possibility of using 3-aminopropylphosphonate as an extracellular pH indicator.

The present inventors have discovered a family of molecules, namelylinear or cyclic aminophosphonates, which are particularly advantageoussince they lead to improved sensitivity in measuring pH and since theymake it possible to cover a whole range of different pH values dependingon the substituents, thus allowing high precision as regards measurementat more acidic or more basic pH values. These molecules are moreoverrelatively non-toxic.

Only some of these aminophosphonates are novel. These novel compoundshave the formula (I.1) or (I.2):

Formula (I.1)

in which:

R represents a (C₁-C₁₈)alkyl or (C₆-C₁₀)aryl group;

R₁ and R₂ independently represent a deuterium atom; a halogen atom; a(C₁-C₁₈)alkyl group optionally substituted with one or more radicalschosen from (C₁-C₆)alkoxy, (C₃-C₁₁)cycloalkyl, halogen, (C₆-C₁₀)aryl andnitro; a (C₆-C₁₀)aryl group optionally substituted with one or moreradicals chosen from (C₁-C₆)alkyl, (C₁-C₆)alkoxy, halogen, nitro and(C₃-C₁₁)cycloalkyl; (C₁-C₈)alkoxy optionally substituted with one ormore radicals chosen from (C₁-C₆)alkoxy, halogen, nitro,(C₃-C₁₁)cycloalkyl and (C₆-C₁₀)aryl; a nitro group; or a(C₃-C₁₁)cycloalkyl group optionally substituted with one or moreradicals chosen from (C₁-C₆)alkyl, (C₁-C₆)alkoxy, halogen and nitro;

R₃ represents a hydrogen or deuterium atom; an n-propyl group or alinear (C₅-C₁₈)alkyl group, optionally substituted with one or moreradicals chosen from: nitro, halogen, (C₁-C₆)alkoxy and(C₃-C₁₁)cycloalkyl; a (C₃-C₁₁)cycloalkyl group optionally substitutedwith one or more radicals chosen from (C₁-C₆)alkyl, (C₁-C₆)alkoxy,halogen and nitro;

and the salts thereof with a pharmaceutically acceptable acid.

Formula (I.2)

in which

R′ represents a hydrogen atom or a (C₁-C₁₈)alkyl or (C₆-C₁₀)aryl group;

R′₁ represents a hydrogen atom; a deuterium atom; a halogen atom; a(C₁-C₁₈)alkyl group optionally substituted with one or more radicalschosen from (C₁-C₆)alkoxy, (C₃-C₁₁)cycloalkyl, halogen, (C₆-C₁₀)aryl andnitro; a (C₆-C₁₀)aryl group optionally substituted with one or moreradicals chosen from (C₁-C₆)alkyl, (C₁-C₆)alkoxy, halogen, nitro and(C₃-C₁₁)cycloalkyl; (C₁-C₁₈)alkoxy optionally substituted with one ormore radicals chosen from (C₁-C₆)alkoxy, halogen, nitro,(C₃-C₁₁)cycloalkyl and (C₆-C₁₀)aryl; a nitro group; or a(C₃-C₁₁)cycloalkyl group optionally substituted with one or moreradicals chosen from (C₁-C₆)alkyl, (C₁-C₆)alkoxy, halogen and nitro;

R′₂ and R′³ together form a divalent radical:

in which the group —C(L₁)(L₂)— is directly linked to the carbon bearingR′₁,

and in which L₁, L₂, L₃ and L₄ represent, independently of each other, ahydrogen atom, a deuterium atom or a (C₁-C₁₈)alkyl or (C₆-C₁₀)arylgroup;

L₅ and L₆ being defined as follows:

when R′₁ represents a hydrogen, halogen or deuterium atom, an optionallysubstituted (C₁-C₁₈)alkoxy group, a nitro group or an optionallysubstituted (C₃-C₁₁)cycloalkyl group, L₅ and L₆ represent, independentlyof each other, a hydrogen atom, a deuterium atom, a (C₁-C₁₈ )alkylgroup, a (C₆-C₁₀)aryl group or a group —P(O)(OR′)₂;

when R′₁ represents an optionally substituted (C₁-C₁₈)alkyl oroptionally substituted (C₆-C₁₀)aryl, either L₅ or L₆ represents ahydrogen atom, and the other represents (C₂-C₁₈)alkyl or (C₆-C₁₀)aryl;

when R′₁ represents methyl, L₁, L₂, L₃, L₄ and L₅ represent a hydrogenatom and R′ represents ethyl, then L₆ is not isopropyl;

and the salts thereof with a pharmaceutically acceptable acid.

The compounds of formula (I.1) are linear phosphonates.

The compounds of formula (I.2) are cyclic phosphonates.

In the context of the invention, the expression “alkyl” means a linearor branched saturated hydrocarbon radical such as methyl, ethyl, propyl,isopropyl, butyl, isobutyl, t-butyl, pentyl, isopentyl, neopentyl,2-methylbutyl, 1-ethylpropyl, hexyl, isohexyl, neohexyl, 1-methylpentyl,3-methylpentyl, 1,1-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl or1-methyl-1-ethylpropyl.

The alkyl radical preferably contains 1 to 10 and better still 1 to 6carbon atoms.

The term “alkoxy” denotes the —O-alkyl radical in which alkyl is asdefined above.

“Halogen” denotes a chlorine, bromine, fluorine or iodine atom, fluorineand chlorine being preferred.

According to the invention, the term “cycloalkyl” denotes saturated,monocyclic or polycyclic, preferably monocyclic or bicyclic,carbocycles.

Cycloalkyls containing 3 to 8 endocyclic carbon atoms are moreparticularly preferred.

Cycloalkyls which may be mentioned are cyclobutyl, cyclopentyl,cyclohexyl, cycloheptyl and cyclooctyl, cyclopentyl and cyclohexyl beingpreferred.

The term “aryl” means a monocyclic or polycyclic, preferably monocyclicor bicyclic, aromatic hydrocarbon radical containing from 6 to 10endocyclic carbon atoms, such as phenyl and naphthyl.

Among the linear compounds of formula (I.1), the ones which arepreferred are those corresponding to one or more of the followingconditions:

1) R₃ is other than a hydrogen atom.

2) R₁ and R₂ are both other than a hydrogen atom.

3) R₃ represents n-propyl or a linear (C₅-C₆)alkyl group, optionallysubstituted with one or more radicals chosen from nitro, halogen,(C₁-C₆)alkoxy and (C₃-C₈)cycloalkyl. R₃ preferably represents n-propylor a linear (C₅-C₆)alkyl group.

4) R₁ and R₂ independently represent a (C₁-C₆)alkyl group optionallysubstituted with one or more radicals chosen from (C₁-C₆)alkoxy,(C₅-C₆)cycloalkyl, halogen, (C₆-C₁₀)aryl and nitro; or alternatively a(C₆-C₁₀)aryl group optionally substituted with one or more radicalschosen from (C₁-C₆)alkyl, (C₁-C₆)alkoxy, halogen, nitro and(C₅-C₆)cycloalkyl; and R represents a (C₁-C₆)alkyl group or a(C₆-C₁₀)aryl group. Advantageously, R₁ and R₂ independently represent(C₁-C₆)alkyl or (C₆-C₁₀)aryl, for example phenyl.

5) R represents a (C₁-C₆)alkyl group or a (C₆-C₁₀ )aryl group.

6) R₁ and R₂ do not represent (C₁-C₁₈)alkyl.

7) At least one from among R₁ and R₂ represents a deuterium atom, ahalogen atom, nitro or optionally substituted (C₁-C₁₈)alkoxy.

Linear compounds of formula (I.1) that are particularly preferred are:

2-(propylamino)-2-(diethoxyphosphoryl)propane

N-[1-phenyl-1-(diethoxyphosphoryl)ethyl]-N-propylamine.

Among the cyclic compounds of formula (I.2), the ones that are preferredare those in which R′ is chosen from (C₁-C₁₈ )alkyl and (C₆-C₁₀ )aryl.

Another group of compounds that are preferred consists of the compoundsof formula (I.2) in which R′₁ represents (C₁-C₆)alkyl or a hydrogen atomand R′₂ and R′₃ together form a radical of formula:

in which L₅ and L₆ are as defined above.

This group of compounds is denoted hereinbelow as the subgroup PC.

When, in this subgroup of preferred compounds, PC, R′₁ represents(C₁-C₆)alkyl, it is preferable for R′₂ and R′₃ together to form adivalent radical:

in which L₅ represents H and L₆ represents (C₆-C₁₀)aryl, for examplephenyl.

When, in the subgroup PC, R′₁ represents a hydrogen atom, it ispreferable for R′₂ and R′₃ together to form a divalent radical:

in which L₅ represents H and L₆ represents —P(O)(OR′)₂ in which R′ is asdefined above.

Among all these compounds, those in which R′ represents a (C₁-C₆)alkylgroup or a (C₆-C₁₀)aryl group are particularly preferred.

A marked preference is given to the following cyclic compounds:

2,5-bis(diethoxyphosphoryl)pyrrolidine; and

2-methyl-2-diethoxyphosphoryl-5-phenyl-pyrrolidine.

The invention covers both the cis and trans isomers of the cyclicderivatives, as well as all the enantiomers and diastereoisomers in thecase in which the compounds of formula (I.1) or (I.2) contain one ormore asymmetric carbons.

According to another of its aspects, the invention relates to the use ofaminophosphonates as pH markers in ³¹P NMR.

More generally, the invention relates to the use, as pH markers, ofcompounds of formula (II.1) or (II.2) or of salts thereof withpharmaceutically acceptable acids:

Formula (II.1)

in which

T₁ and T₂ independently represent a group —R or —OR;

R represents a (C₁-C₁₈)alkyl or (C₆-C₁₀)aryl group;

R₁ and R₂ independently represent a hydrogen atom; a deuterium atom; ahalogen atom; a (C₁-C₁₈)alkyl group optionally substituted with one ormore radicals chosen from (C₁-C₆)alkoxy, (C₃-C₁₁)cycloalkyl, halogen,(C₆-C₁₀)aryl and nitro; a (C₆-C₁₀)aryl group optionally substituted withone or more radicals chosen from (C₁-C₆)alkyl, (C₁-C₆)alkoxy, halogen,nitro and (C₃-C₁₁)cycloalkyl; (C₁-C₁₈)alkoxy optionally substituted withone or more radicals chosen from (C₁-C₆)alkoxy, halogen, nitro,(C₃-C₁₁)cycloalkyl and (C₆-C₁₀)aryl; a nitro group; a group —P(O)(OR)₂;or a (C₃-C₁₁)cycloalkyl group optionally substituted with one or moreradicals chosen from (C₁-C₆)alkyl, (C₁-C₆)alkoxy, halogen and nitro;

R₃ represents a hydrogen or deuterium atom; a (C₁-C₁₈)alkyl groupoptionally substituted with one or more radicals chosen from nitro,halogen, (C₁-C₆)alkoxy, (C₆-C₁₀)aryl and (C₃-C₁₁)cycloalkyl, andoptionally bearing a group —P(O)(OR)₂ in position 1; a(C₃-C₁₁)cycloalkyl group optionally substituted with one or moreradicals chosen from (C₁-C₆)alkyl, (C₁-C₆)alkoxy, halogen and nitro, a(C₆-C₁₀)aryl group optionally substituted with one or more radicalschosen from (C₁-C₆)alkyl, (C₆-C₁₀)aryl, (C₁-C₆)alkoxy, nitro, halogenand (C₃-C₁₁)cycloalkyl;

p represents 0 or 1;

A represents a divalent radical —CR₄R₅— in which: R₄ and R₅ have themeanings given above for R₁ and R₂ with the exclusion of —P(O)(OR)₂;

it being understood that the said compound does not contain more thantwo groups —P(O)(OR)₂.

Formula (II.2)

in which T′₁ and T′₂ independently represent (C₁-C₁₈)alkyl;(C₆-C₁₀)aryl; or a group —OR′; R′ represents a hydrogen atom or a(C₁-C₁₈)alkyl or (C₆-C₁₀)aryl group; R′₁ represents a hydrogen atom; adeuterium atom; a halogen atom; a (C₁-C₁₈)alkyl group optionallysubstituted with one or more radicals chosen from (C₁-C₆)alkoxy,(C₃-C₁₁)cycloalkyl, halogen, (C₆-C₁₀)aryl and nitro; a (C₆-C₁₀)arylgroup optionally substituted with one or more radicals chosen from(C₁-C₆)alkyl, (C₁-C₆)alkoxy, halogen, nitro and (C₃-C₁₁)cycloalkyl;(C₁-C₁₈)alkoxy optionally substituted with one or more radicals chosenfrom (C₁-C₆)alkoxy, halogen, nitro, (C₃-C₁₁)cycloalkyl and (C₆-C₁₀)aryl;a nitro group; a group —P(O)(OR′)₂; or a (C₃-C₁₁)cycloalkyl groupoptionally substituted with one or more radicals chosen from(C₁-C₆)alkyl, (C₁-C₆)alkoxy, halogen and nitro; R′₂ and R′₃ togetherform a divalent radical:

in which the group —C(L₁)(L₂)— is directly linked to the carbon bearingR′₁ and in which

L₁, L₂, L₃ and L₄ represent, independently of each other, a hydrogenatom; a deuterium atom; a (C₁-C₁₈)alkyl group optionally substitutedwith one or more radicals chosen from (C₁-C₆)alkoxy, (C₃-C₁₁)cycloalkyl,halogen, (C₆-C₁₀)aryl and nitro; a (C₆-C₁₀)aryl group optionallysubstituted with one or more radicals chosen from (C₁-C₆)alkyl,(C₁-C₆)alkoxy, halogen, nitro and (C₃-C₁₁)cycloalkyl; (C₁C₁₈)alkoxyoptionally substituted with one or more radicals chosen from(C₁-C₆)alkoxy, halogen, nitro, (C₃C₁₁)cycloalkyl and (C₆-C₁₀)aryl; anitro group; a group —P(O)(OR′)₂; or a group (C₃-C₁₁)cycloalkyloptionally substituted with one or more radicals chosen from(C₁-C₆)alkyl, (C₁-C₆)alkoxy, halogen and nitro; and L₅ and L₆ represent,independently of each other, a hydrogen atom; a deuterium atom; a(C₁-C₁₈)alkyl group optionally substituted with one or more radicalschosen from (C₁-C₆)alkoxy, (C₃-C₁₁)cycloalkyl, halogen, (C₆-C₁₀)aryl andnitro; a (C₆-C₁₀)aryl group optionally substituted with one or moreradicals chosen from (C₁-C₆)alkyl, (C₁-C₆)alkoxy, halogen, nitro and(C₃-C₁₁)cycloalkyl; (C₁-C₁₈)alkoxy optionally substituted with one ormore radicals chosen from (C₁-C₆)alkoxy, halogen, nitro,(C₃-C₁₁)cycloalkyl and (C₆-C₁₀)aryl; a nitro group; a group —P(O)(OR′)₂;

or a (C₃-C₁₁)cycloalkyl group optionally substituted with one or moreradicals chosen from (C₁-C₆)alkyl, (C₁-C₆)alkoxy, halogen and nitro; ora group —P(O)(OR′)₂;

p′ represents 0 or 1;

A′ represents a divalent radical —CR′₄R′₅— in which R′₄ and R′₅ have themeanings given above for R′₁ with the exclusion of —P(O)(OR′)₂,

it being understood that the said compound does not contain more thantwo groups —P(O)(OR′)₂.

The compounds of formula (I.1) above are a subgroup of the compounds ofthe formula (II.1).

One of the meanings of R₃ is n-propyl or linear (C₅-C₁₈)alkyl,optionally bearing a group —P(O)(OR)₂ in position 1. This means that thecarbon atom of the n-propyl group or of the (C₅-C₁₈)alkyl group which isdirectly linked to the nitrogen atom can bear a group —P(O)(OR)2, asillustrated below:

Similarly, the compounds of formula (I.2) above are a subgroup of thecompounds of formula (II.2).

According to one preferred embodiment of the invention, the pH markerused is a linear phosphonate corresponding to one or more of theconditions (i) to (xi) below:

i) a compound of formula (II.1) in which R₃ is other than a hydrogenatom;

ii) a compound of formula (II.1) in which R₁ and R₂ are both other thana hydrogen atom;

iii) a compound of formula (II.1) in which p represents 0;

iv) a compound as defined in iii) for which R₃ represents a (C₁-C₆)alkylgroup optionally substituted with one or more radicals chosen fromnitro, halogen, (C₁-C₆)alkoxy, (C₆-C₁₀)aryl and (C₃-C₈)cycloalkyl, andoptionally bearing a group —P(O)(OR)₂ in position 1;

v) a compound as defined in iii) for which R₁ and R₂ independentlyrepresent a (C₁-C₆)alkyl group optionally substituted with one or moreradicals chosen from (C₁-C₆)alkoxy, (C₅-C₆)cycloalkyl, halogen,(C₆-C₁₀)aryl and nitro; a (C₆-C₁₀)aryl group optionally substituted withone or more radicals chosen from (C₁-C₆)alkyl, (C₁-C₆)alkoxy, halogen,nitro and (C₅-C₆)cycloalkyl or alternatively a group —P(O)(OR)₂; and Rrepresents a (C₁-C₆)alkyl group or a (C₆-C₁₀)aryl group;

vi) a compound as defined in iii) for which R₃ represents (C₁-C₆)alkyl;R₁ and R₂ independently represent (C₁-C₆)alkyl or phenyl optionallysubstituted with one or more (C₁-C₆)alkyl, (C₁-C₆)alkoxy, halogen, nitroand (C₅-C₆)cycloalkyl radicals; or alternatively a group —P(O)(OR)₂;

vii) a compound chosen from2-(propylamino)-2-(diethoxyphosphoryl)propane,N-[1-phenyl-1-(diethoxyphosphoryl)ethyl]-N-propylamine andN-[1,1-bis(diethoxyphosphoryl)methyl]-N-tert-butylamine;

viii) a compound of formula (II.1) in which p represents 1; R₃represents (C₁-C₆)alkyl; A represents —CR₄R₅—; R₁, R₂, R₄ and R₅ areindependently chosen from a hydrogen atom and a (C₁-C₆)alkyl group; andR represents a (C₁-C₆)alkyl group or a (C₆-C₁₀)aryl group;

ix) N-[(1-methyl-2-diethoxyphosphoryl)ethyl]-N-n-butylamine; and ethyl[1-tert-butylamino-2,2-(dimethyl)propyl][methyl]phosphinate;

x) a compound of formula (II.1) in which T₁ represents —OR and T₂represents —R;

xi) a compound of formula (II.1) in which T₁ and T₂ represent —OR.

According to another preferred embodiment of the invention, a cyclicphosphonate of formula (II.2) is used, corresponding to one or more ofthe conditions (xii) to (xvii) below:

xii) compound of formula (II.2) in which R′ is other than a hydrogenatom;

xiii) a compound of formula (II.2) in which R′₁ represents a hydrogenatom, a (C₁-C₆)alkyl group, (C₆-C₁₀)aryl group or a group —P(O)(OR′)₂and R′₂ and R′₃ together form a radical of formula:

 in which L₅ and L₆ are as defined for formula (II.2);

xiv) a compound as defined in xi) in which L₅ and L₆ are chosenindependently from a hydrogen atom, a (C₁-C₆)alkyl group, a (C₆-C₁₀)arylgroup or a group —P(O)(OR′)₂, R₁ representing a (C₁-C₆)alkyl group, a(C₆-C₁₀)aryl group or a hydrogen atom;

xv) a compound chosen from:

—N-[(1-methyl-2-diethoxyphosphoryl)ethyl]-N-n-butylamine;

2-methyl-2-diethoxyphosphorylpyrrolidine;

2,2-bis(diethoxyphosphoryl)pyrrolidine;

2,2-bis(diisopropoxyphosphoryl)pyrrolidine;

trans-2,5-bis(diethoxyphosphoryl)pyrrolidine;

2-phenyl-2-diethoxyphosphorylpyrrolidine;

2-methyl-2-diethoxyphosphoryl-5-phenylpyrrolidine; and

ethyl 2-methylpyrrolidin-2-yl methyl phosphinate;

xvi) a compound of formula (II.2) in which T′₁ represents —OR′ and T′₂is (C₁-C₁₈)alkyl or (C₆-C₁₀)aryl;

xvii) a compound of formula (II.2) in which T′₁ and T′₂ represent —OR′.

The use of the preferred compounds of formula (I.1) and (I.2) aboveforms another preferred embodiment of the invention.

In a particularly advantageous manner, the compounds used as pH markersare those of formula (II.2) comprising two functions —P(O)(OR′)₂ (whichform a preferred subgroup of the invention), and more specifically thecompounds of formula (II.2) in which:

either R₁ represents —P(O)(OR′)₂;

or L₅ or L₆ represents —P(O)(OR′)₂.

The following preparation processes allow the synthesis of the compoundsof formulae (II.1) and (II.2) and thus of the compounds of formulae(I.1) and (I.2).

A) The compounds of formula (II.2) for which p′ represents 0; T′₁ andT′₂ represent —OR′; and R′₂ and R′₃, taken together, form a radical:

in which L₁ to L₆ are as defined in formula (II.2) and comprise in theirmolecule only one function —P(O)(OR′)₂, can be prepared by reacting acompound of formula (III):

in which R′₁, L₁, L₂, L₃, L₄, L₅ and L₆ are as defined for formula(II.2) and X represents a halogen atom such as a chlorine, bromine oriodine atom, with a compound of formula (IV):

in which R′ is as defined for formula (II.2), in the presence of NH₃.

The reaction conditions depend on the nature of the reagents of formulae(III) and (IV) and may readily be determined by a person skilled in theart. The reaction is generally carried out in a solvent, for example apolar protic solvent. The solvent is advantageously ethanol. Thetemperature is generally maintained between room temperature and thereflux temperature of the solvent.

The compounds of formulae (III) and (IV) are commercially availablecompounds or compounds that can readily be prepared by a person skilledin the art starting with commercially available compounds.

This process is illustrated in patent application FR 93/08906.

B) As a variant, the compounds of formula (II.2) targeted in paragraphA) above and for which R′₁ does not represent H, can be prepared by theaction of a pyrroline of formula (V):

in which R′₁, L₁, L₂, L₃, L₄, L₅ and L₆ are as defined above for (II.2),on a compound (IV) of formula:

in the presence of a Lewis acid of formula MX_(n) in which X is ahalogen atom, M is an element chosen from B, Al, Fe, Ga, Sb, Sn, As, Znand Hg and n is an integer between 2 and 5 whose value corresponds tothe valency of the element M in the compound MX_(n). MX_(n) preferablyrepresents BF₃, this acid generally being used in the form of itsBF₃—Et₂O complex.

The reaction can be carried out at room temperature in a polar aproticsolvent such as an ether, and for example tetrahydrofuran or diethylether. A 10 mol % to 50 mol % excess of the compound of formula (IV) ispreferably reacted with the pyrroline (V).

The pyrroline of formula (V) can be prepared according to the reactionscheme below:

in which R′₁ and L₁ to L₆ are as defined for the formula (II), M₀represents an alkali metal and in particular sodium or potassium; Xrepresents a halogen atom; and Ar represents a C₆-C₁₀ aromatic groupoptionally substituted with a C₁-C₆ alkyl group.

According to this reaction scheme, the compound of formula (III) isreacted with an alkali metal azide of formula M₀N₃ in which M₀represents an alkali metal. M₀N₃ is preferably NaN₃ and the reaction iscarried out in a polar aprotic solvent in the presence of an ammoniumchloride such as tetrabutylammonium chloride. An example of a solventwhich may be mentioned is dimethoxyethane.

As a guide, 1 to 3 molar equivalents of sodium azide are reacted withthe derivative of formula (III), preferably 1 to 2 molar equivalents.

The reaction of the resulting compound of formula (VI) with thetriarylphosphine of formula P(Ar)₃ in which Ar denotes an optionallysubstituted (C₆-C₁₀) aromatic radical is generally carried out in apolar aprotic solvent, preferably diethyl ether. Ar advantageouslyrepresents phenyl. The reaction is stoichiometric. The process ispreferably performed in the presence of an excess of P(Ar)₃.

The cyclization takes place at room temperature after adding a solvent,for instance a hydrocarbon such as pentane to the reaction medium. Thisreaction is continued for the time required, occasionally for 36 hours;reaction for 5 to 15 hours is generally required. Whatever the case, aperson skilled in the art may vary the temperature and the solvent, in amanner which is known per se, to improve the reaction kinetics.

C) The compounds of formula (II.2) targeted in paragraph A) above, inwhich L₆ represents a hydrogen atom, can also be prepared by reducingthe corresponding nitrones of formula (VIII):

A reducing agent which will be used, for example, is tributyltin hydrideor NaHTe. In this respect, a person skilled in the art will refer to D.H. R. Barton et al. (1985), Tetrahedron Letters, 26, 4603.

Patent application FR 93/08906 describes a general method for preparingthe compounds of formula (VIII).

As a variant, the compounds of formula (VIII) in which L₂, L₄ and L₆represent a hydrogen atom and R′₁ is other than a hydrogen atom can beprepared by carrying out the following sequence of reaction steps:

in which R′₁, L₁, L₃, L₅ and R′ are as defined for (II).

The reaction of compound (IX) with compound (X) is advantageouslycarried out in a polar aprotic solvent such as acetonitrile in thepresence of a base such as triethylamine, pyridine or4-dimethylaminopyridine in catalytic amount. The reaction temperature isgenerally between room temperature and the reflux temperature of thesolvent.

The resulting compound of formula (XI) is reacted with excess zinc inthe presence of acetic acid at a temperature of between 0° C. and 100°C., to give the expected compound (VIII).

This reaction can be carried out in a solvent. In this case, a polarprotic solvent such as ethanol will preferably be chosen.

The amount of zinc is advantageously between 1 and 5 molar equivalentsrelative to compound (IX), preferably between 1 and 3 equivalents.

The compound of formula (X) is readily prepared (i) by reacting acetylchloride with a trialkyl phosphite of formula P(OR′)₃ according to theArbuzov method, and then (ii) reacting the resulting dialkyl2-oxoethylphosphonate with hydroxylamine and (iii) oxidizing theresulting oxime into 1-nitroethylphosphonate (X). The latter oxidationreaction is described in particular in Zon et al., Synthesis, 1984,661-663 and uses meta-chloroperbenzoic acid as oxidizing agent. This setof steps is reported below:

CH₃COCl+P(OR′)₃→CH₃COP(O)(OR′)₂+R′Cl

CH₃COP(O)(OR′)₂+NH₂OH→CH₃C═N(OH)—P(O)(OR′)₂

CH₃C═N(OH)—P(O)(OR′)₂→CH₃CH(NO₂)—P(O)(OR′)₂ in which R′ is as definedfor formula (II.2).

D) The compounds of formula (II.2) for which p′ represents 0; T′₁ andT′₂ represent —OR′; and R′₂ and R′₃, taken together, form a radical:

in which L₁ to L₆ are as defined in formula (II.2) and R′₁ represents—P(O)(OR′)₂, can be prepared from the corresponding 2-oxopyrrolidines offormula (XII):

in which L₁ to L₆ are as defined for formula (II).

According to this process, the 2-oxopyrrolidine (XII) is successivelyreacted with′ the appropriate trialkyl phosphite of formula P(OR′)₃ inwhich R′ is as defined for formula (II), under an inert atmosphere, at atemperature ranging between −10° C. and room temperature, and then witha phosphoryl halide of formula P(O)X₃ in which X represents a halogenatom, at this same temperature. According to a preferred embodiment, thereaction mixture is kept stirring while optionally allowing thetemperature to rise to room temperature for 1 to 10 hours. The reactionmixture is then treated in a second step with ammonium hydroxide. Atleast two molar equivalents (relative to the amount of compound (XII))of P(OR′)₃ and of P(O)X₃ are required for this reaction.

E) The compounds of formula (II.2) in which p′ represents 0; T′₁ and T′₂represent —OR′; and R′₂ and R′₃, taken together, form a radical:

in which L₁ to L₅ and R′₁ are as defined in formula (II.2), and L₆represents —P(O)(OR′)₂, can be prepared according to following reactionscheme:

According to one preferred embodiment, the reaction of (XIII) with (IV)is carried out in the presence of a large excess of compound (XIII):from 5 to 20 molar equivalents of compound (XIII) give satisfactoryyields. The reaction takes place in the presence of ammonia at atemperature between room temperaature and 100° C., preferably between20° C. and 70° C. In a second step, the resulting compound of formula(XIV) is reacted with compound (IV), preferably with an excess of thecompound of formula (IV). The molar ratio of compound (XIV) to compound(IV) is generally between 10:1 and 2:1, preferably between 5:1 and 2:1.

F) The linear compounds of formula, (II.1) in which p represents 0 andT₁ and T₂ represent —OR are prepared simply by addition of an amine offormula (XVI):

R₃—NH₂  (XVI)

in which R₃ is as defined for the formula (II), to a ketone of formula(XVII):

in which R₁ and R₂ are as defined for formula (II), in the presence of acompound of formula (IV):

in which R is as defined for formula (II). This reaction isstoichiometric but can be carried out in the presence of an excess ofthe amine (XVI) and/or of the phosphite (IV). The molar ratio of theamine (XVI) to the ketone (XVII) preferably ranges between 0.2 and 2 andbetter still between 0.8 and 1.8. The molar ratio of the phosphite (IV)to the ketone (XVII) preferably ranges between 0.3 and 2 and betterstill between 0.5 and 1.5.

The reaction can be carried out without solvent or in the presence of asolvent. Preferably, the reagents are used as solvents. The temperatureis maintained between 200° C. and 500° C. The reaction is advantageouslycarried out at room temperature.

According to one specific embodiment, the amine (XVI) is reacted at roomtemperature with the ketone (XVII) in the presence of an alkali metalsulphate and a strong acid, such as the system Na₂SO₄/HCl. In this case,a mixture of the ketone (XVII) and the amine (XVI) is prepared and theNa₂SO₄/HCl system is added thereto. After a reaction time of between 1and 72 hours, the phosphite (IV) is added to the reaction mixture.

From 0.5 to 2 equivalents of alkali metal sulphate and a catalyticamount of the strong acid are preferably used. The alkali metal ischosen from sodium, potassium, lithium and caesium, sodium beingpreferred.

G) The linear compounds of the formula (II.1) in which p represents 0;T₁ and T₂ represent —OR; and R₃ comprises a group —P(O)(OR)₂ can beprepared by adapting the process described by Von K. Issleib in Z.anorg. allg. Chem. 444, 249-255 (1978).

This process is more particularly suitable for synthesizing thecompounds of formula (XVIII):

in which R is as defined for formula (II.1) and R_(a) and R_(b)independently represent a (C₁-C₁₈)alkyl group optionally substitutedwith one or more radicals chosen from nitro, halogen, (C₁-C₆)alkoxy,(C₆-C₁₀)aryl and (C₃-C₈)cycloalkyl; or alternatively R_(a) and R_(b)form, together with the carbon atom which bears them, a(C₃-C₈)cycloalkyl.

According to this process, an aldehyde of formula R_(a)R_(b)CH—CHO istreated with ammonia. Thermolysis of the resulting compound gives thedimer of formula:

R_(a)R_(b)C═CH—N═CH—CHR_(a)R_(b)

which is reacted at a temperature of between 15° C. and 35° C. with twomolar equivalents of the phosphite of formula (IV):

in which R is as defined above.

The compounds of formula (XVIII) in which R_(a) and R_(b) are chosenindependently from (C₁-C₆)alkyl or form, together with the carbon atomwhich bears them, a (C₅-C₈)cycloalkyl group, are prepared in particularby this method.

H) The linear compounds of formula (II.1) in which p represents 0; T₁and T₂ represent —OR; and either R₁ or R₂ represents —P(O)(OR)₂, can beprepared by the action of two molar equivalents of a suitable trialkylphosphite of formula P(OR)₃ in which R is as defined for (II.1), on aformamide of formula (XIX):

R₃—NH—COH  (XIX)

in the presence of phosphorus oxychloride POCl₃ at a temperature ofbetween −15° C. and 0° C., preferably between −10° C. and 0° C.

Although 2 molar equivalents of the trialkyl phosphite are generallysufficient, it is possible to use a slight molar excess of phosphite.Thus, the molar ratio of the trialkyl phosphite to compound (XIX)preferably ranges between 2.5 and 2, preferably between 2.2 and 2.

The reaction is generally carried out by adding POCl₃ to a solutionconsisting of the mixture of the trialkyl phosphite and the formamide,maintained at −5° C.

The molar amount of POCl₃ used in this reaction ranges between 2 and 2.5mol per 1 mol of the compound (XIX). The molar ratio of POCl₃ to thetrialkyl phosphite preferably ranges between 1 and 1.3.

I) The linear compounds of formula (II.1) in which p represents 1 andeither R₁ or R₂ represents a hydrogen atom, can be prepared by carryingout the process which follows:

In a first step, the trialkyl phosphite of formula P(OR)₃ in which R isas defined for formula (II.1) is reacted with a chloride of formula (XX)

at a temperature of between 80° C. and 200° C. This reaction isdescribed more specifically in N. D. Dawson et al., J. Am. Chem. Soc.,74, 5312-5314, 1952. A person skilled in the art will known how to adaptthe reaction conditions to the nature of the various reagents placed incontact. This reaction is preferably carried out in the absence ofsolvent at a temperature of from 140° C. to 180° C. The productresulting from this first step, of formula:

is reacted with an amine of formula R₃—NH₂ in the presence of a hydride,preferably in the presence of NaBH(OAc)₃ and of a C₁-C₄ alkylcarboxylicacid such as acetic acid. This reaction is preferably carried out in thepresence of a halogenated hydrocarbon solvent (such as dichloroethane)at a temperature of between 15° C. and 35° C., for example at roomtemperature (22° C.). After this reaction, the desired compound offormula (II) is obtained.

The reactions used in these two steps are stoichiometric. Equivalentmolar amounts of phosphite P(OR)₃ and of chloride (XX), and respectivelyof the compound (XXI) and of the amine R₃—NH₂, will thus be placed incontact. In the second step, the molar ratio of the alkylcarboxylic acidto the compound (XXI) may range between 1 and 5, preferably between 1and 2. Moreover, the molar ratio of the hydride to the compound (XXI)will be adjusted to between 1 and 1.5, preferably between 1 and 1.2.

J) The compounds of general formula (II.2) in which p′ represents 1; T′₁and T′₂ represent —OR′; and R′₂ and R′₃, taken together, form a radical

in which L₁ to L₆ are as defined in formula (II.2) and comprising intheir molecule only one function —P(O)(OR′)₂, can be prepared:

(i) by reacting a compound of formula (XXII)

in which L₁ to L₆, R′₁, R′₄ and R′₅ are as defined above and Prorepresents a protecting group for an amine function, for example abenzyloxycarbonyl group, with a phosphoryl derivative of the formulaP(OR′)₃ in which R′ is as defined for formula (II.2); and

(ii) by deprotecting the secondary amine function of the productobtained from the first step (i).

This synthetic method is described and illustrated in particular inFR-A-2 707 990.

The protecting functions which can be used to protect the endocyclicnitrogen of the pyrrolidine ring are those conventionally used inorganic chemistry. A person skilled in the art will refer, for example,to Protective Groups in Organic Synthesis, Greene T. W. and Wuts P. G.M., published by John Wiley and Sons, 1991. This book also describes thecorresponding deprotection methods.

The compounds of formula (XXII) can be prepared in two steps from thecompounds of formula (XXIII)

in which L₁ to L₆, R′₁ and Pro are as defined above for compound (XXII).

In a first step, a compound (XXIII) is reacted with mercury diacetate;the second step, which consists in treating the resulting productsuccessively with potassium iodide and iodine, leads directly to thecorresponding compound of formula (XXII).

The compounds of formula (XXIII) are readily prepared by a personskilled in the art using conventional organic chemistry processes,starting with commercially available compounds.

K) The compounds of formula (II.2) as defined in E) in the case where L₅and R′₁=H can also be prepared according to the following scheme:

Step 1: Synthesis of Pyrrolidine-2,5-diphosphonic Acid

Step 2: Esterification

L) The compounds of formula (II.2) in which p′=0 and at least one fromamong T′₁ and T′₂ represents (C₁-C₁₈)alkyl or (C₆-C₁₀)aryl can beprepared according to the following synthetic scheme:

in which T′₁, T′₂, R′₁ and L₁ to L₆ are as defined in formula (II.2)above; alk represents (C₁-C₆)alkyl and GP represents a leaving group,preferably a halogen atom such as chlorine.

The reaction of the phosphinate XXXa with the silyl derivative XXXI isstoichiometric. The molar ratio of compound XXXI to compound XXXa thusgenerally ranges between 1 and 1.5, preferably between 1 and 1.2.

This reaction is carried out in the presence of a base, preferably anorganic base such as a tertiary amine. Suitable bases areN-methylmorpholine, triethylamine, tributylamine, diisopropylamine,dicyclohexylamine, N-methylpiperidine, pyridine,4-(1-pyrrolidinyl)pyridine, N,N-dimethylaniline and N,N-diethylaniline.

The reaction of XXXa with XXXI is preferably carried out in a polarsolvent such as a halogenated aliphatic hydrocarbon such asdichloromethane, carbon tetrachloride or dichloroethane.

The reaction temperature is preferably maintained between −20° C. and10° C. and better still between −5° C. and 5° C.

In a second step, the compound XXXII obtained is reacted with theappropriate pyrroline of formula XXXIII. This reaction is preferablycarried out in situ, without intermediate isolation of the compoundXXXII obtained above.

A molar ratio of the pyrroline XXXIII to the silyl derivative XXXII offrom 1 to 1.5 and preferably from 1 to 1.2 is generally suitable.

The reaction of XXXIII with XXXII is generally carried out in a polarsolvent such as a halogenated aliphatic hydrocarbon as defined above.

This process is particularly suitable for preparing the compounds (II.2)above in which T′₁ represents (C₁-C₁₈)alkyl or (C₆-C₁₀)aryl and T′₂represents —OR′.

M) The compounds of formula (II.1) in which p=0 and at least one fromamong T₁ and T₂ represents —R can be prepared in accordance with thefollowing reaction scheme:

in which R₁, R₂, R₃, T₁ and T₂ are as defined above in formula (I.1).

This reaction can be carried out in the absence of solvent or in thepresence of an inert solvent capable of dissolving the reagents XXXb andXXXIV.

A suitable temperature is a temperature of between 15° C. and 80° C.,preferably between 30° C. and 50°0 C.

The compounds of formulae XXXa and XXXb are prepared conventionallyaccording to the standard methods of organic chemistry.

By way of illustration, the phosphinate XXXa (or XXXb respectively) [inwhich T₁ (or T′₁ respectively) represents —OR (or OR′ respectively) andT₂ (or T′₂ respectively) represents R (or (C₁-C₁₈)alkyl or (C₆-C₁₀)arylrespectively)] can be obtained by reacting a halophosphite of formula(alk₀)(T₁)Phal₁ (or (alk₀) (T′₁)Phal₁ respectively) in which hal₁represents a halogen atom, preferably chlorine, and alk₀ represents(C₁-C₆)alkoxy, with a magnesium reagent of formula T₂Mghal₂ (orT′₂Mghal₂ respectively) in which hal₂ is a halogen atom, preferably aniodine atom, followed by reaction of the resulting compound with anammonium halide, for example ammonium chloride. This reaction sequencecan be represented schematically as follows:

The imine XXXIV is prepared conventionally according to the knownmethods of organic chemistry and, for example, by the action of an amineon an aldehyde.

The compounds XXXI are commercially available or readily prepared by aperson skilled in the art starting with commercially availablecompounds.

This process is particularly suitable for preparing the compounds (II.1)above in which T₁ represents —R and T₂ represents —OR.

The compounds of formulae (II.1) and (II.2) (or (I.1) and (I.2)respectively) can be isolated in the form of their salts with an organicor inorganic acid, for example picric acid, oxalic acid, tartaric acid,mandelic acid or camphorsulphonic acid. However, the physiologicallyacceptable salts are preferred, such as the hydrochloride, hydrobromide,sulphate, hydrogen sulphate, dihydrogen phosphate, maleate, fumarate,2-naphthalenesulphonate or para-toluenesulphonate.

The originality of the phosphonates of formula (II.2) lies mainly intheir rigid cyclic structure.

Among the compounds of formula (II.2), those for which R′ is other thana hydrogen atom are preferred.

All the compounds of formula (II.1) (which are linear phosphonates) aresuch that R is other than a hydrogen atom.

When R′ in formula (II.2) and R in formula (II.1) are other than ahydrogen atom, the function(s) —P(O)(OR′)₂, or —P(O)(OR)₂ respectively,are in the form of phosphonate groups.

For all the compounds of the invention, the chemical shift of thephosphorus, in ³¹P NMR, depends on the pH. More specifically, thechemical shift of the phosphorus varies greatly for pH values close tothe pKa of the compound of formula (II.1) or (II.2) being investigated.For pH values that are far from the pKa value, the chemical shift of thephosphorus tends towards a constant.

The variation in the chemical shift of the phosphorus (δ) as a functionof the pH can be represented schematically in the following way:

The inventors have found, surprisingly, that when all the functions—P(O)(OR′)₂ and —P(O)(OR)₂ in the compounds of the invention are in theform of phosphonates (R′≠H and R≠H), then the difference Δδ=δ_(b)−δ_(a)is particularly large. Now, the greater the value of Δδ, the greater thepH measurement sensitivity. Thus, the compounds of formulae (II.1) and(II.2) in which R, or R′ respectively, is other than a hydrogen atom areparticularly sensitive and reliable pH markers.

Whereas the pH markers of the prior art generally show. a difference Δδof 2 to 3 ppm, the compounds of the invention of formulae (II.1) and is(II.2) in which R′ is other than a hydrogen atom have a Δδ which is fourtimes as large.

Depending on the nature of the substituents R₁, R₂ and R₃ (or R′₁, R′₂and R′₃ respectively), the pKa value of the compound of formula (II.1)(or (II.2) respectively) varies. In point of fact, the pKa value dependson the electron-withdrawing or electron-donating effect of thesesubstituents.

When R′ represents a hydrogen atom, the family of compounds of formula(II.2) has a rather narrow pKa distribution, compared with the pKadistribution obtained with the corresponding family of compounds offormula (II.2) for which R′≠H.

Similarly, the compounds of the invention of formula (II.1) have a broaddistribution of pKa values, compared with the corresponding family ofcompounds combining the compounds of formula (II.3) below:

in which R₁, R₂ and R₃ are as defined for formula (II.1) and R″represents H or is as defined above for R in formula (II.1).

More specifically, the compounds of the invention have pKa values ofbetween 2 and 9.

It will be noted that the diphosphoryl compounds, and in particular thecyclic diphosphoryl compounds of formula (II.2), also give a greater Δδvariation.

In order to demonstrate the ability of the compounds of the formulae(I.1), (I.2), (II.1) and (II.2) above to function as pH-markers, thevariation in the chemical shift of the ³¹P NMR peak as a function of thepH was studied. The results obtained allowed titration curves to beplotted.

The attached FIGS. 1 to 8 are titration curves obtained using thefollowing compounds:

FIG. 1: Titration curve for 2-methyl-2-diethoxyphosphorylpyrrolidine:compound 1

FIG. 2: Titration curve for 2,2-bis(diethoxyphosphoryl) pyrrolidine:compound 2

FIG. 3: Titration curve for 2,2-bis(diisopropoxyphosphoryl) pyrrolidine:compound 3

FIG. 4: Titration curve for2,5-bis(diethoxyphosphoryl)-2,5-dimethylpyrrolidine: compound 4

FIG. 5: Titration curve for 2-phenyl-2-diethoxyphosphorylpyrrolidine:compound 5

FIG. 6: Titration curve forN-propyl-N-[1-phenyl-1-diethoxyphosphorylethyl]amine: compound 6

FIG. 7: Titration curve for 2-propylamino-2-diethoxyphosphorylpropane:compound 7

FIG. 8: Titration curve forN-[(1-methyl-2-diethoxyphosphoryl)ethyl]-N-n-butylamine.

Each measurement was taken at 37° C. using a 5 mM solution of the testcompound in a phosphate buffer on a 400 MHz NMR spectroscopy machine.

Table 1 below reports the pKa values measured using the curves plottedfor each of the test compounds.

TABLE 1 Compound 1 2 3 4 5 6 7 8 PKa 6.75 3.47 3.90 2.45 5.73 5.67 6.778.64

The pKa values measured show that compounds 1 to 7 allow pH measurementsover a very broad pH range. More generally, by modifying the nature ofthe substituents R, R₁, R₂, R₃, R′₁, R′₂, R′₃ and R′ of the compounds offormulae (I.1), (I.2), (II.1) and (II.2), it is possible to provide pHmarkers that are particularly sensitive in different pH zones, down tothe most acidic values, a given compound ensuring good measurementaccuracy only in the pH zone surrounding its pKa. This presents acertain advantage over the known pH markers, since the known compoundsdo not make it possible to study the acidic compartments of cells.

The compounds of formulae (I.1), (I.2), (II.1) and (II.2) are thus pHmarkers that are particularly advantageous, offering greater accuracy inthe measurement of intracellular pH.

It is confirmed that the diphosphoryl compounds give higher NMRsensitivity, thus making it possible to reduce the concentration ofmarker required.

For comparative purposes, three additional experiments were carried outin order to compare the sensitivity of compound 1 and inorganicphosphate.

The variation in the phosphorus-31 chemical shift was studied as afunction of the pH: the measurements were taken at 22° C., or 37° C.respectively, on a 400 MHz NMR machine using 5 mM solutions of the testcompound in a phosphate buffer.

The results obtained are reported in FIGS. 9 to 11:

FIG. 9: Titration curve for compound 1, at 22° C.

FIG. 10: Titration curve for inorganic phosphate, at 22° C.

FIG. 11: Titration curve for inorganic phosphate, at 37° C.

These curves show that when the pH varies between 4 and 10, the chemicalshift of the inorganic phosphate peak in ³¹P NMR varies between 0.062and 2.741 at 37° C. (FIG. 11) and between 0.086 and 2.481 at 22° C.(FIG. 10), i.e. by an amplitude of between 2.679 and 2.395 according tothe experimental conditions. On the other hand, in the case of compound1, with the pH varying between 4 and 10, the chemical shift of thephosphorus peak in ³¹P NMR varies between 23.185 and 32.834 at 37° C.(FIG. 1) and between 23.137 and 32.779 at 22° C. (FIG. 9), i.e. by anamplitude of between 9.649 and 9.642 according to the experimentalconditions.

The pKa values of these various compounds are reported in Table 2 below.

TABLE 2 Compound Compound 1 Pi Pi Temperature 22° C. 22° C. 37° C. PKa7.06 6.75 6.65

These results demonstrate the higher measurement sensitivity obtainedwith the compounds of formulae (II.1) and (II.2).

Thus, the superiority of the compounds of formulae (I.1), (I.2), (II.1)and (II.2) over inorganic phosphate is incontrovertible.

The compounds of formulae (II.1) and (II.2) are on the whole particuarlynon-toxic and penetrate into myocardial or liver cells (in sufficientamount to be observed but in a sufficiently small amount to remainnon-toxic), thus allowing a measurement of the pH values ofintracellular and extracellular media. Better still, in the case of theliver which possesses very acidic (pH 5) vesicles, the marker2-diethoxyphosphoryl-2-methylpyrrolidine allows three measurements atthe same time: extracellular medium, cytosolic medium (in the case of2-diethoxyphosphoryl-2-methylpyrrolidine, variations in the chemicalshift of the phosphorus as a function of the intracellular acidosis areobserved, in the course of ischaemia, which are, for example, fullycorrelated with those of the intracellular Pi) and intravesicular medium(vesicles whose function is yet to be determined).

Accordingly, the compounds of formulae (II.1) and (II.2) are verypowerful tools for enabling a better understanding of the functioning ofdifferent cellular organells and the proton fluxes which ensure theirfunctioning.

The following abbreviations have been used in the examples:

iPr: isopropyl n-Bu: n-butyl CHCl₃: chloroform bp: boiling point δ:chemical shift

s: singlet; d: doublet; t: triplet; dd: doubled doublet; q: quartet;sext.: sextet; m: multiplet: J: coupling constant.

EXAMPLES Example 1 Synthesis of Compound 1 of Formula

A mixture of 2-methylpyrroline 4 (10.9 g; 0.13 mol) and diethylphosphite (21.7 g; 0.16 mol) is kept stirring for 7 days at roomtemperature. After addition of 150 ml of HCl solution (1N), the mixtureis then washed with CH₂Cl₂ (2×80 ml). The aqueous phase is then basifiedwith sodium carbonate and the product is extracted with CHCl₃ (3×100ml). The organic phase is dried over sodium sulphate and concentratedunder reduced pressure. Compound 1 is obtained in the form of acolourless oil (27.30 g; 95%).

IR (KBr, cm⁻¹): 3315, 1235, 1055, 1037, 958; ³¹P NMR (C₆D₆) δ (ppm)29.6; (CDCl₃) δ (ppm) 29.9; ¹H NMR (C₆D₆, 100 MHz) δ (ppm) 1.10 (t,J=7.0 Hz, 6H); 1.28 (d, J=15 Hz, 3H); 1.20-1.80 (m, 3H); 2.00-2.50 (m,1H); 2.70-3.00 (m, 2H); 3.70-4.50 (m, 4H); ¹³C NMR (C₆D₆) δ (ppm) 16.63;16.72; 19.53; 26.08 (d, J=4.5 Hz); 35.18 (d, J=2.5 Hz); 50.35 (d,J=150.7 Hz); 62.05; 62.30 (d, J=7.2 Hz). Elemental analysis: calculatedfor C₉H₂₀NO₃P: C: 48.89; H: 9.11; N: 6.33; Found: C: 48.49; H: 9.15; N:6.24.

Example 2 Synthesis of Compound 2 of Formula

Phosphorus oxychloride (40 ml; 0.44 mol) is added over 1 h 15 min at −5°C. to a solution of 2-pyrrolidinone (18.5 g; 0.22 mol) and triethylphosphite (9.42 mol). The reaction medium is stirred for 5 hours at roomtemperature and then poured onto a mixture of ice (300 g) and 32%aqueous ammonia (300 ml). The aqueous phase is extracted withdichloromethane (4×100 ml) and the organic extracts are evaporated underreduced pressure to give a yellow oil. The oil is dissolved in 100 ml ofdichloromethane and 200 ml of water are added, followed by addition of37% hydrochloric acid to pH 1. The aqueous phase is washed withdichloromethane (4×50 ml). Sodium hydroxide and sodium carbonate areadded to pH 10 and the aqueous phase is extracted with dichloromethane(4×50 ml). The organic phase is dried over sodium sulphate, filtered andthen evaporated under reduced pressure to give the gem-bisphosphonate.33.4 g of the expected product (47% yield) are thus obtained.

¹H NMR (400 MHz, C₆D₆) δ (ppm) 1.10 (t, 6H J=7.1 Hz, —O—CH₂—CH ₃); 1.11(t, 6H, J=7.1 Hz, —O—CH₂—CH ₃); 1.69 (q, 2H, J=6.8 Hz; J_(Ha-Hb)=7.2 Hz,HN—CH₂—CH ₂—CH₂—C); 2.42 (n, 2H, J_(Ha-Hb)=7.2 Hz, J_(P-H)=17.7 Hz,HN—CH₂—CH_(2(b))—CH_(2(a))—C); 2.88 (t, 2H, J=6.5 Hz, HN—CH₂—CH₂—CH₂—C);4.17 (m, 8H, —O—CH ₂CH₃); ¹³C (100 MHz); (C₆D₆) δ (ppm) 16.5 (t,J_(C-P)=7.2 Hz, CH ₃—CH₂—OP); 16.6 (t, J_(C-P)=5.5 Hz, CH₃—CH₂—O—P—),26.5 (t, J_(C-P)=3.1 Hz, HN—CH₂—CH₂—CH₂—C); 31.2 (t, J_(C-P)=3.0 Hz,HN—CH₂—CH₂—CH₂—C); 47.7 (t, J_(C-P)=4.0 Hz, HN—CH₂—CH₂—CH₂—C); 62.7 (t,J_(C-P)=3.6 Hz, CH₃—CH₂—O—P—); 62.8 (t, J_(C-P)=151.8 Hz,HN—CH₂—CH₂—CH₂—C); 63.4 (t, J_(C-P)=5.3 Hz, CH₃—CH₂—O—P—); ³¹P (40 MHz;CDCl₃) δ 22.5 ppm; IR (without solvent): 3480 (NH); 2982, 2932; 2909;2869; 1456; 1392; 1243; (P═O); 1164 (P—OC₂H₅); 1044; 968; 794; 732; 645;580; 536 cm⁻¹ bp: 140° C. (8 Pa) pKa: 3.5 Rf: 0.39 (acetone); 0.43(dichloromethane/ethanol: 19/1).

Example 3 Synthesis of Compound 3 of Formula

Using a procedure similar to that of Example 2, starting with phosphorusoxychloride, 2-pyrrolidinone and triisopropyl phosphite, 37.2 g of thetitle compound (45% yield) are obtained.

¹H NMR (400 MHz; C₆D₆) δ (ppm) 1.22 (d, 6H J_(H-H)=6.1 Hz, —O—CH(CH₃)₂); 1.27 (d, 6H, J_(H-H)=6.4 Hz, —O—CH(CH ₃)₂); 1.28 (d, 6H,J_(H-H)=6.3 Hz, —O—CH(CH ₃)₂); 1.31 (d, 6H, J_(H-H)=6.2 Hz,—O—CH(CH₃)₂); 1.75 (q, 2H, J=6.8 Hz, J_(H-H)=6.9 Hz, HN—CH₂—CH ₂—CH₂—C);2.37 (tt, 2H, J_(H-H)=7.3 Hz, J_(P-H)=17.7 Hz, HN—CH₂—CH₂—CH ₂—C); 2.95(t, 2H, J=6.5 Hz, HN—CH ₂—CH₂—CH₂-C); 4.87 (m, 1H, —O—CH(CH₃)₂); 5.00(m, 1H, —O—CH(CH₃)₂); ³C (100 MHz; C₆D6) δ (ppm) 23.8 (t, ³J_(C-P)=6.7Hz, —O—CH(CH₃)₂); 24.0 (t, ³J_(C-P)=6.4 Hz, —O—CH(CH₃)₂); 24.4 (s,—O—CH(CH₃)₂); 24.7 (s, —O—CH(CH₃)₂); 26.4 (t, ³J_(C-P)=3.3 Hz,HN—CH₂—CH₂—CH₂—C); 31.0 (t, ²J_(C-P)=3.4 Hz, HN—CH₂—CH₂—CH₂—C); 47.7 (t,³J_(C-P)=4.6 Hz, HN—CH₂—CH₂—CH₂—C); 63.1 (t, ²J_(C-P)=151.1,NH—CH₂—CH₂—CH₂—C); 70.8 (t, ²J_(C-P)=6.7 Hz, —O—CH(CH₃)₂); 71.7 (t,²J_(C-P)=6.2 Hz, —O—CH(CH₃)₂); ³¹P (40 MHz; CDCl₃) δ 21.2 ppm; Elementalanalysis: calculated for C₁₆H₃₅NO₆P₂:

C H N Calculated (%) 48.12 8.83 3.51 Found (%) 48.21 8.80 3.51

Example 4 Synthesis of Compound 5 of Formula

a) 2-Phenylpyrroline

14 g of chlorobutyrophenone (0.078 mol) and 1.5 equivalents of sodiumazide (7.6 g; 0.117 mol) in 60 ml of dimethoxyethane are mixed with 0.5g of tetrabutylammonium chloride in a 250 ml two-necked round-bottomedflask equipped with a condenser, under an inert atmosphere.

The mixture is maintained at 75° C. on an oil bath for 16 hours. Thereaction mixture is filtered through Celite. After extraction with ethylether and evaporation of the solvent under reduced pressure, 16.2 g ofcrude product are obtained in the form of a dark oil. This oil was notpurified.

The crude product is taken up with 200 ml of anhydrous ethyl ether in a500 ml three-necked round-bottomed flask, to which is added 20.4 g oftriphenylphosphine (0.777 mol) in several portions. This results in anevolution of nitrogen, which may be observed by installing a guard tube.100 ml of pentane are then added and the mixture is left at roomtemperature for 12 hours under magnetic stirring. The triphenylphosphineoxide precipitated is filtered off on a sinter funnel. After evaporationof the solvents under reduced pressure, 12.5 g of crude product areobtained. The pyrroline is recrystallized from pentane.

¹H NMR: (100 MHz, CDCl₃, TMS) 7.9-7.8 (m, 2H); 7.4-7.3 (m, 3H); 4.1-4(tt, 2H); 2.9-2.8 (tt, 2H); 2.1-1.9 (m, 2H).

b) 2-Phenyl-2-diethoxyphosphorylpyrrolidine

1.2 equivalents of diethyl phosphite are added to 12.5 g of thepyrroline prepared in a) over 24 hours and 1 ml of the diethylether/trifluoroborane catalyst in ethyl ether, with magnetic stirring.

The reaction mixture is treated with 0.1 N hydrochloric acid solutionuntil a pH of 1 is obtained. The aqueous phase thus obtained is treatedwith sodium hydroxide solution until a pH of 8 is obtained. Thetreatment is completed by addition of Na₂CO₃ and the mixture is thensaturated with NaCl. The resulting mixture is extracted again withdichloromethane (4×30 ml). The organic phase is dried over MgSO₄. Afterevaporation of the solvents under reduced pressure, 9.8 g of crudeproduct (45% yield) are obtained in the form of a yellow oil. Thephosphite is evaporated under reduced pressure. The pyrrolidine ispurified by chromatography on silica with acetone/pentane (1/3) aseluent, in a yield of 60%.

³¹P (C₆D₆) δ 26.9 ppm; ¹H NMR: (400 MHz, C₆D₆): 0.92 (3H, t, OCH₂CH₃,³J_(HH)=7.07 Hz); 0.97 (3H, t, OCH₂CH₃, ³J_(HH)=7.07 Hz); 1.45-1.34 (1H,m, CH); 1.69-1.58 (1H, m, CH); 2.24-2.15 (1H, m, CH); 2.49 (1H, broad s,NH); 2.63-2.51 (1H, m, CH); 2.79 (1H, dd, CH): 3.0-2.94 (1H, m, CH);3.85-3.67 (2H, m, OCH₂CH₃); 3.94-3.87 (2H, m, OCH₂CH₃); 7.11-7.06 (1H,m, H_(ar)); 7.24-7.20 (2H, m, H_(ar)); 7.83-7.79 (2H, m, H_(ar)). ¹³C(100.6, MHz, C₆D₆): 16.78 and 16.83 (OCH₂ CH₃); 25.94 (CH₂(3), J=9.05Hz); 37.33 (CH₂(2) ); 47.22 (CH₂(4), J=9.05 Hz); 62.66 and 63.04(OCH₂CH₃, J 7.0 Hz): 67.7 (Ph—C ₁—P, J=149.9 Hz); 127.41 (CH(8), J=3.0Hz); 128.36 (CH(6), J=3.0 Hz); 128.49 (CH(7), J=4.01 Hz); 142.7 C(5).

Example 5 Synthesis of Compound 6 of Formula

3.45 g of benzophenone (0.029 mol; 1 eq.) 2.8 g of n-propylamine (0.047mol; 1.6 eq.), 2 drops of concentrated hydrochloric acid and 4.3 g ofNa₂SO₄ (0.03 mol; 1 eq.) are introduced into. a reactor under magneticstirring. After stirring for 3 days at room temperature, 4.83 g ofdiethyl phosphite (0.035 mol: 1.2 eq.) are added to the reactionmixture. After stirring for 10 days at room temperature, an acid-basetreatment is carried out in order to obtain the expected compound. Theyield is 51%.

Example 6 Synthesis of Compound 7 of Formula

24 g of acetone (0.4 mol; 1 eq.), 28 g of diethyl phosphite (0.2 mol;0.5 eq.) and 25 g of n-propylamine (0.42 mol; 1 eq.) are introduced intoa reactor under magnetic stirring. The mixture is left to react for 3days at room temperature and an acid-base treatment is carried out inorder to obtain the expected compound, which has a boiling point of 70°C. at 0.06 mbar. Yield=66%.

Example 7 Synthesis of2,5-bis(Diethoxyphosphoryl)-2,5-dimethylpyrrolidine (Compound 4)

Step 1

Preparation of Diethyl (2,5-dimethyl-1-pyrrolin-5-yl)phosphonate

Hexane-2,5-dione (25.68 g; 0.225 mol) and diethyl phosphite (22.48 g;0.163 mol) are placed in a 500 ml three-necked round-bottomed flaskequipped with a condenser and a magnetic stirrer. Ammonia is thenbubbled through at a temperature of 35° C. for 21 hours. The reactionmixture is then treated with 0.1 M HCl solution to pH=1, after which itis extracted with diethyl ether. The aqueous phase obtained is treatedby addition of NaHCO₃ to pH=10. The amine is salted out by adding NaCland a large excess of Na₂CO₃ with stirring, in the presence of diethylether. The aqueous phase is then extracted with diethyl ether, afterwhich the organic phase is dried over anhydrous Na₂SO₄. After filtrationand evaporation under vacuum, the expected compound is obtained in theform of a yellow oil (12.95 g; 34%).

³¹P NMR (40.53 MHz, CDCl₃) 27.59 ppm ¹H NMR: (100 MHz, CDCl₃); 1.32 ppm(6H; t; J_(H-H)=6.98 Hz; CH ₃—CH₂); 1.48 ppm (3H; d; J_(H-P)=16.29 Hz;CH ₃—C—P); 2.07 ppm (3H; d; J_(H-P)=4.65 Hz; CH ₃—C═N); between 2.5 and2.8 ppm (4H; m; CH ₂—CH₂); 4.15 ppm (4H; dq; J_(H-H) =J _(H-P)=6.98 Hz;CH₃—CH₂); ¹³C NMR: (25.18 MHz, CDCl₃): 16.39 ppm (d; J_(C-P)=7.25 Hz;CH₃—CH₂); 19.64 ppm (d′ J_(C-P)=2.9 Hz; CH₃—C═N); 23.48 ppm (s;CH₃—C—P); 32.37 ppm (d; J_(C-P)=3.73 Hz; CH₂—CH₂); 39.59 ppm (s;CH₂—CH₂); 62.45 ppm (d; J_(C-P)=7.43 Hz; CH₂—CH₃); 177.32 ppm (d;J_(C-P)=13.62 Hz; C═N)

Step 2

Preparation of 2,5-bis(Diethoxyphosphoryl)-2,5-dimethylpyrrolidine

Diethyl 2,5-dimethyl-1-pyrrolin-5-yl)phosphonate prepared in step 1above (4.87 g; 0.0208 mol) is placed in a 250 ml two-neckedround-bottomed flask and diethyl phosphite (5.89 g; 0.043 mol) is addeddropwise thereto. The reaction is stirred at room temperature for 5days. The reaction mixture is treated with 0.1 M HCl solution to pH=1and is then extracted with diethyl ether. The aqueous phase obtained is:treated by addition of NaHCO₃ to pH=10. The amine is salted out byadding NaCl and a large excess of Na₂CO₃ with stirring, in the presenceof diethyl ether. The aqueous phase is then extracted with diethylether, after which the organic phase is dried over anhydrous Na₂SO₄.After filtration and evaporation under vacuum, the title compound isobtained in the form of an orange oil (3.41 g, 44%).

³¹P NMR (40.53 MHz, CDCl₃): 29.23 ppm; ¹H NMR: (100 MHz, CDCl₃); 1.33ppm (6H; t; J_(H-H)=6.78 Hz; CH ₃—CH₂); 2.16 ppm (6H; d; J_(H-P)=1.13Hz; CH₃—C—P): between 2 and 2.5 ppm (4H; m; CH ₂—CH₂); 4.18 ppm (4H; dq;J_(H-H)=J_(H-P)=6.78 Hz; CH₃—CH ₂) ¹³C NMR: (25.18 MHz, CDCl₃): 16.34ppm (d; J_(C-P)=6.57 Hz; CH₃—CH₂); 24.42 ppm (d; J_(C-P)=4.45 Hz;CH₃—C—P); 34.26 ppm (s; CH₂); 54.55 ppm (d; J_(C-P)=150 Hz; N—C—P);61.95 ppm (d; J_(C-P)=6.82 Hz; CH₂—CH₃)

Example 8 Synthesis of the Compound of Formula

Using a procedure similar to that of Example 2, starting with phosphorusoxychloride, 2-pyrrolidinone and tri-n-butyl phosphite, 23.2 g of thetitle compound (47% yield) are obtained.

¹H NMR: (400 MHz; C₆D₆) δ (ppm) 0.82 (t, 6H, J=7.4 Hz, —O—CH₂—CH₂—CH₂—CH₃); 0.83 (t, 6H, J=7.4 Hz, —O—CH₂—CH₂—CH₂—CH ₃); 1.32 (sext. 4H, J=7.3Hz, —O—CH₂—CH₂—CH ₂—CH₃); 1.33 (sext. 4H, J: 7.5 Hz, —O—CH₂—CH₂—CH₂—CH₃); 1.56 (m, 8H, —O—CH₂—CH ₂—CH₂—CH₃); 1.76 (q, 2H, J=6.9 Hz,J_(H-H)=7.2 Hz, HN—CH₂—CH ₂—CH₂—C); 2.49 (n, 2H, J_(Ha-Hb)=7.3 Hz,J_(P-H)=17.8 Hz, HN—CH₂—CH_(2(b))—CH_(2(a))—C); 2.97 (t, 2H, J=6.5 Hz,HN—CH ₂—CH₂—CH₂—C); 4.22 (m, 4H, —O—CH ₂—CH₂—CH₂—CH₃); 4.27 (m, 4H,—O—CH ₂—CH₂—CH₂—CH₃); 5.30 (s, 1H, HN—); ¹³C (100 MHz; C₆D₆) δ (ppm)14.1 (CH₃—CH₂—CH₂—CH₂—O—P); 19.4 (CH₃—CH₂—CH₂—CH₂—O—P); 26.8 (t,J_(C-P)=3.2 Hz, HN—CH₂—CH₂—CH₂—C); 31.5 (t, J_(C-P)=3.0 Hz,HN—CH₂—CH₂—CH₂—C); 33.4 (t, J_(C-P)=2.7 Hz, CH₃—CH₂—CH₂—CH₂—O—P); 33.5(t, J_(C-P)=2.7 Hz, CH₃—CH₂—CH₂—CH₂—O—P); 48.1 (t, J_(C-P)=4.3 Hz,HN—CH₂—CH₂—CH₂—C); 63.2 (t, J_(C-P)=152.0 Hz, HN—CH₂—CH₂—CH₂—C); 67.1(t, J_(C-P)=3.5 Hz, CH₃—CH₂—CH₂—O—P); 67.8 (t, J_(C-P)=3.2 Hz,CH₃—CH₂—CH₂—CH₂—O—P) ³¹P (40 MHz; CDCl₃): δ 22.7 ppm.

Example 9 Preparation of the Compound of Formula

Triethyl phosphite (23 g; 0.14 mol) and N-tert-butylformamide (7.5 g;0.073 mol) are placed in a 250 ml two-necked round-bottomed flask. Thecompounds are mixed together at room temperature for a few minutes. Anice bath with salt is installed and at −5 °C., 23 g of POCl₃ (i.e. 0.15mol) are added. The addition takes 1 hour. The reaction is then leftstirring at room temperature for 5 hours. The solution gradually turnsorange. The crude mixture is then poured into a beaker containing 150 gof ice and 150 ml of 32% aqueous ammonia solution. The aqueous phase isextracted with twice 150 ml of dichloromethane. 100 ml of water areadded, followed by dropwise addition of 37% HCl solution until the pH=1.After extraction with 4 times 20 ml of dichloromethane, the aqueousphase is recovered and NaHCO₃ is added thereto until the pH=10. Afterextraction with diethyl ether, drying over Na₂SO₄ and evaporation of thesolvent, the title compound is obtained in the form of a yellowish oil(9.63 g; 56%).

³¹P NMR (40.53 MHz, CDCl₃); 20.01 ppm; ¹H NMR (200 MHz, CDCl₃); 1.12 ppm(9H; s; CH₃—C); 1.35 ppm (12 H; t; J_(H-H)=6 Hz: CH ₃—CH₂); 2.77 ppm(1H; m; CH—P); 4.13-4.29 ppm (8 H; m; CH ₂) ¹³C NMR (50.32 MHz, CDCl₃);16.10 ppm (m, CH₃—CH₂); 29.20 ppm (S; CH₃—C); 48.95 ppm (t;J_(C-P)=148.5 Hz; CH—P); 51.70 ppm (t; J_(C-P)=10.5 Hz; C—N); 62.54 ppm(d; J_(C-P)=4 Hz; CH₂); 62.61 ppm (d; J_(C-P)=3.5 Hz; CH₂); 63.13 ppm(d; J_(C-P)=3.2 Hz); 63.21 ppm (d; J_(C-P)=3.85 Hz); Elemental analysis:calculated for C₁₃H₃₁NO₆P₂; C: 43.45; H: 8.70; N: 3.90 Found: C: 43.12;H: 8.86; N: 3.58.

Example 10 Synthesis of the Compound of Formula

Step 1

A mixture of 29 g (0.314 mol; 1 eq.) of chloroacetone and 52 g oftriethyl phosphite (0.314 mol; 1 eq.) is refluxed for 4 hours at 160° C.

6 g of diethylacetylmethane phosphonate are obtained after distillationunder vacuum (1 mmHg; t=98° C.) (yield: 10%).

Step 2

A mixture of 1 g of the β-acetophosphonate obtained in step 1 (5.15×10⁻³mol/l eq.) is stirred for 8 hours under an inert atmosphere, at roomtemperature, with 0.40 g of n-butylamine (1 eq.), 1.64 g of NaBH(OAc)₃(7.73×10⁻³ mol, i.e. 1.5 eq.) and 0.33 g of acetic acid (5.7×10⁻³mol/1.1 eq.) in 10 ml of dichloroethane.

Step 3

5 ml of water are added to the reaction mixture. The solution isacidified with 35% HCl solution. The impurities are extracted with 3×10ml of dichloromethane. The aqueous phase is then basified with NaOHsolution. The expected compound is then extracted with 3×10 ml ofdichloromethane. The organic phases are dried over MgSO₄ and thenevaporated. 0.7 g of the title compound is obtained (55% yield).

Example 11 Synthesis of 2,5-bis(Diethoxyphosphoryl)pyrrolidine

Acetyl chloride (7.8 g; 0.1 mol) is added dropwise over 10 minutes to amixture of freshly distilled butanedial (0.05 mol), acetamide (14.7 g;0.25 mol) and acetic acid (50 ml) maintained at 0° C. with vigorousstirring.

The solution is then stirred for 12 hours at room temperature. Themixture is then cooled to 0° C. and phosphorus trichloride PCl₃ (8.8 g;0.1 mol) is introduced dropwise with vigorous stirring over 15 minutes.The reaction mixture is then refluxed on a water bath for 1 hour, afterwhich it is evaporated under reduced pressure. The oily residue is takenup in 100 ml of 12 M hydrochloric acid and refluxed for 12 hours, thenevaporated under reduced pressure on a water bath. The residue istreated with 60 ml of methanol and the ammonium chloride precipitatethus obtained is filtered off and then washed with two successivefractions of 20 ml of methanol. The methanol fractions are combined andthen evaporated under reduced pressure, after which the residue obtainedis dissolved in a minimum amount of water. The aqueous solution ispassed through an ion exchange column (Dowex 50*2-100, H⁺ form) andeluted with water. The pyrrolidine-2,5-diphosphonic acid is obtained ina yield of 39%. Reference: I. van Assche et al., 51991) Eur. J. Med.Chem. 26, 505-515.

This diphosphonic acid can be esterified using a trialkyl orthoformateHC(OR)₃ in the presence of para-toluenesulphonic acid (R=CH₃ or C₂H₅).For general esterification methods, a person skilled in the art willrefer to the reference article: U.S. Schollkopf et al., (1985) LiebigsAnn. Chem. 555-559.

Example 12 Synthesis of Ethyl 2-Methylpyrrolidin-2-ylmethylphosphinateof Formula

0.8 g (7.4 mmol) of ethyl methylphosphinate of formula (CH₃)(H)P(O)OC₂H₅and 488 mg (8 mmol) of triethylamine are dissolved in 20 ml of anhydrousmethylene chloride and cooled to 0° C. 0.87 g (8 mmol) of trimethylsilylchloride is then added. The formation of the intermediateP(OSiMe₃)(OEt)(Me) is virtually instantaneous. 0.63 g (7.6 mmol) of2-methylpyrroline of formula:

is added slowly. The mixture is then left stirring at room temperature.After 20 hours, the reaction monitoring by ³¹P NMR shows no furtherappreciable progress. The mixture is then hydrolysed with 20 ml ofaqueous 10% hydrochloric acid solution and then purified by acid-basetreatment. After extraction with methylene chloride, drying overanhydrous Na₂SO₄ and evaporation under vacuum, 1.1 g of crude productare obtained. Purification on a preparative plate gives 760 mg of thepure expected compound. Yield: 53%.

NMR:

Diestereoisomer 1:

¹H (400 MHz; CDCl₃); δ (ppm) 4.07 (m, O—CH₂—CH₃); 2.8 to 3.1 (m, CH₂—N); 1.5 to 2.2 (m, CH ₂—CH ₂—CH₂—N); 1.43 (d; J=12.9 Hz; CH₃—C_(quat).); 1.29 (d; J=14.75 Hz; CH ₃—P); 1.28 (t; J=7 Hz; O—CH₂—CH₃). ¹³C (100.61 MHz; CDCl₃); δ (ppm) 60.68 (d; J=7.2 Hz; O—CH₂—CH₃);60.61 (d; J=118 Hz; C _(quat).); 47.3 (d; J=8 Hz; CH₂—C_(quat).); 34.06(d; J=4 Hz; CH₂—CH₂ —C_(quat).): 26.08 (CH₂—N); 23.13 (d; J=8.2 Hz;CH₃—C_(quat).); 16.77 (O—CH₂—CH₃); 9.62 (d; J=87.5 Hz; CH₃—P). ³¹P(40.53 MHz; CDCl₃): δ (ppm) 57.52.

Diestereoisomer 2:

¹H (400 MHz; CDCl₃); δ (ppm) 4.07 (m, O—CH₂—CH₃); 2.8 to 3.1 (m, CH₂—N); 1.5 to 2.2 (m, CH ₂—CH ₂—CH₂—N); 1.42 (d; J=12.8 Hz; CH₃—C_(quat).); 1.27 (t; J=7 Hz; O—CH₂—CH ₃); 1.26 (d; J=1.26 Hz; CH ₃—P).¹³C (100.61 MHz; CDCl₃); δ (ppm) 60.62 (d; J=7.1 Hz; O—CH₂—CH₃); 60.53(d; J=118.8 Hz; Cquat.); 47.25 (d; J=7.9 Hz; CH₂—C_(quat).); 33.52 (d;J=4.2 Hz; CH₂—CH₂—C_(quat).); 26.04 (CH₂—N); 23.31 (d; J=7.7 Hz;CH₃—C_(quat).); 16.69 (O—CH₂—CH₃); 9.44 (d; J=87 Hz; CH₃—P) ³¹P (40.53MHz; CDCl₃): δ (ppm) 57.30.

What is claimed is:
 1. A pH marker of formula:

in which: R is selected from the group consisting of (C₁-C₁₈)alkyl and(C₆-C₁₀)aryl; R₁ and R₂ are independently selected from the groupconsisting of a deuterium atom; a halogen atom; a (C₁-C₁₈)alkyl groupunsubstituted or substituted with one or more radicals selected from thegroup consisting of (C₁-C₆)alkoxy, (C₃-C₁₁)cycloalkyl, halogen,(C₆-C₁₀)aryl and nitro; a (C₆-C₁₀)aryl group unsubstituted orsubstituted with one or more radicals selected from the group consistingof (C₁-C₆)alkyl, (C₁-C₆)alkoxy, halogen, nitro and (C₃-C₁₁)cycloalkyl;(C₁-C₁₈)alkoxy substituted with one or more radicals selected from thegroup consisting of (C₁-C₆)alkoxy, halogen, nitro, (C₃-C₁₁)cycloalkyland (C₆-C₁₀)aryl; a nitro group; and a (C₃-C₁₁)cycloalkyl groupunsubstituted or substituted with one or more radicals selected from thegroup consisting of (C₁-C₆)alkyl, (C₁-C₆)alkoxy, halogen and nitro; R₃is selected from the group consisting of a deuterium atom; a linear(C₅-C₁₈)alkyl group unsubstituted or substituted with one or moreradicals selected from the group consisting of nitro, halogen,(C₁-C₆)alkoxy and (C₃-C₁₁)cycloalkyl; and a (C₃-C₁₁)cycloalkyl groupunsubstituted or substituted with one or more radicals selected from thegroup consisting of (C₁-C₆)alkyl, (C₁-C₆)alkoxy, halogen and nitro; itbeing understood that: when R represents ethyl and R₁ and R₂ representpropyl, then R₃ does not represent dodecyl; and when R representsiso-C₈H₁₇ and R₁ and R₂ represent methyl, then R₃ does not representhexyl; or the salts thereof with a pharmaceutically acceptable acid. 2.Compound according to claim 1, of formula (I.1) in which R₃ represents alinear (C₅-C₆)alkyl group, unsubstituted or substituted with one or moreradicals selected from the group consisting of nitro, halogen,(C₁-C₆)alkoxy and (C₃-C₈)cycloalkyl.
 3. Compound according to claim 1,of formula (I.1) in which R₁ and R₂ are independently selected from thegroup consisting of a (C₁-C₆)alkyl group unsubstituted or substitutedwith one or more radicals selected from the group consisting of(C₁-C₆)alkoxy, (C₅-C₆)cycloalkyl, halogen, (C₆-C₁₀)aryl and nitro; and a(C₆-C₁₀)aryl group unsubstituted or substituted with one or moreradicals selected from the group consisting of (C₁-C₆)alkyl,(C₁-C₆)alkoxy, halogen, nitro and (C₅-C₆)cycloalkyl; and R is selectedfrom the group consisting of a (C₁-C₆)alkyl group and a (C₆-C₁₀)arylgroup.
 4. Compound according to claim 1, of formula (I.1) in which R₃represents a linear (C₅-C₆)alkyl group; and R₁ and R₂ are independentlyselected from the group consisting of (C₁-C₆)alkyl and phenylunsubstituted or substituted with one or more radicals selected from thegroup consisting of (C₁-C₆)alkyl, (C₁-C₆)alkoxy, halogen, nitro and(C₅-C₆)cycloalkyl.
 5. A pH marker of formula:

in which: R′ is selected from the group consisting of a hydrogen atom; a(C₁-C₁₈)alkyl and a (C₆-C₁₀)aryl group; R′₁ is selected from the groupconsisting of a hydrogen atom; a deuterium atom; a halogen atom; a(C₁-C₁₈)alkyl group optionally substituted with one or more radicalsselected from the group consisting of (C₁-C₆)alkoxy, (C₃-C₁₁)cycloalkyl,halogen, (C₆-C₁₀)aryl and nitro; a (C₆-C₁₀)aryl group unsubstituted orsubstituted with one or more radicals selected from the group consistingof (C₁-C₆)alkyl, (C₁-C₆)alkoxy, halogen, nitro and (C₃-C₁₁)cycloalkyl;(C₁-C₁₈)alkoxy unsubstituted or substituted with one or more radicalsselected from the group consisting of (C₁-C₆)alkoxy, halogen, nitro,(C₃-C₁₁)cycloalkyl and (C₆-C₁₀)aryl; a nitro group; or a(C₃-C₁₁)cycloalkyl group unsubstituted or substituted with one or moreradicals selected from the group consisting of (C₁-C₆)alkyl,(C₁-C₆)alkoxy, halogen and nitro; R′₂ and R′³ together form a divalentradical:

in which the group —C(L₁)(L₂)— is directly linked to the carbon bearingR′₁, and in which L₁, L₂, L₃ and L₄ are, independently of each otherselected from the group consisting of a hydrogen atom, a deuterium atom,a (C₁-C₁₈)alkyl and a (C₆-C₁₀)aryl group; L₅ and L₆ being defined asfollows: when R′₁ is selected from the group consisting of a hydrogen,halogen or deuterium atom, an unsubstituted or substituted(C₁-C₁₈)alkoxy group, a nitro group and an unsubstituted or substituted(C₃-C₁₁)cycloalkyl group, L₅ and L₆ are, independently of each other,selected from the group consisting of a hydrogen atom, a deuterium atom,a (C₁-C₁₈)alkyl group, a (C₆-C₁₀)aryl group and a group —P(O)(OR′)₂;when R′₁ is selected from the group consisting of an unsubstituted orsubstituted (C₁-C₁₈)alkyl and an unsubstituted or substituted(C₆-C₁₀)aryl, either L₅ or L₆ represents a hydrogen atom, and the otheris selected from the group consisting of (C₂-C₁₈)alkyl and (C₆-C₁₀)aryl;it being understood that when L₁, L₂, L₃, L₄, and either L₅ or L₆represent a hydrogen atom, then: if R′ and R′₁ represent H, then L₆ isother than H; if R′ represents (C₁-C₃)alkyl and R′₁ represents methyl,then L₆ is neither ethyl nor isopropyl; if R′ represents (C₁-C₃)alkyland R′₁ represents H, then L₆ is not hexyl; or the salts thereof with apharmaceutically acceptable acid, wherein if R′ represents (C₁-C₃)alkyland R′₁ represents H, then L₆ is neither hexyl nor methyl.
 6. Compoundaccording to claim 5, of formula (I.2) in which R′ is selected from thegroup consisting of (C₁-C₁₈)alkyl and (C₆-C₁₀)aryl.
 7. Compoundaccording to claim 5, of formula (I.2) in which R′₁ is selected from thegroup consisting of (C₁-C₆)alkyl and a hydrogen atom and R′₂ and R′₃together form a radical of formula:

either L₅ or L₆ represents a hydrogen atom and the other represents(C₂-C₁₈)alkyl or (C₆-C₁₀)aryl, it being understood that when R′₁represents methyl, then either L₅ or L₆ represents H and the other isother than ethyl or isopropyl.
 8. Compound according to claim 5, offormula (I.2) in which R′₁ represents (C₁-C₆)alkyl, R′₂ and R′₃ togetherform a divalent radical:

in which L₅ represents H and L₆ represents phenyl; and R′ is selectedfrom the group consisting of a (C₁-C₆)alkyl group, a (C₆-C₁₀)aryl groupand a hydrogen atom.
 9. Compound according to claim 5, of formula (I.2)in which R′₁ represents a hydrogen atom and R′₂ and R′₃ together form adivalent radical:

in which L₅ represents H and L₆ represents —P(C)(OR′)₂; and R′ isselected from the group consisting of a (C₁-C₆)alkyl group, a(C₆-C₁₀)aryl group and a hydrogen atom.
 10. Method of pH measurement byNMR spectroscopy comprising: measuring pH with a pH-marker in ³¹P NMR,wherein said pH marker is a compound of formula:

 in which: T₁ and T₂ independently represent a group —R or —OR; R isselected from the group consisting of a (C₁-C₁₈)alkyl and (C₆-C₁₀)arylgroup; R₁ and R₂ are independently selected from the group consisting ofa hydrogen atom; a deuterium atom; a halogen atom; a (C₁-C₁₈)alkyl groupunsubstituted or substituted with one or more radicals selected from thegroup consisting of (C₁-C₆)alkoxy, (C₃-C₁₁)cycloalkyl, halogen, (C₆-C₁₀)aryl and nitro; a (C₆-C₁₀)aryl group unsubstituted or substituted withone or more radicals selected from the group consisting of (C₁-C₆)alkyl,(C₁-C₆)alkoxy, halogen, nitro and (C₃-C₁₁) cycloalkyl, (C₁-C₁₈)alkoxyunsubstituted or substituted with one or more radicals selected from thegroup consisting of (C₁-C₆)alkoxy, halogen, nitro, (C₃-C₁₁)cycloalkyland (C₆-C₁₀)aryl; a nitro group; a group —P(O)(OR)₂, and a(C₃-C₁₁)cycloalkyl group or substituted with one or more radicalsselected from the group consisting of (C₁-C₆)alkyl, (C₁-C₆)alkoxy,halogen and nitro; R₃ is selected from the group consisting of ahydrogen and a deuterium atom; a (C₁-C₁₉)alkyl group unsubstituted orsubstituted with one or more radicals selected from the group consistingof nitro, halogen, (C₁-C₆)alkoxy, (C₆-C₁₀)aryl and (C₃-C₁₁)cycloalkyl,and optionally bearing a group —P(O) (OR)₂ in position 1; a(C₃-C₁₁)cycloalkyl, group unsubstituted or substituted with one or moreradicals selected from the group consisting of (C₁-C₆)alkyl,(C₆-C₁₀)alkoxy, nitro, halogen and (C₃-C₁₁)cycloalkyl; p represents 0 or1; A represents a divalent radical —CR₄R₅— in which R₄ and R₅ have themeanings given above for R₁ and R₂ with the exclusion of —P(O) (OR)₂; itbeing understood that the said compound does not contain more than twogroups —P(O)(OR)₂; or a salt thereof with pharmaceutically acceptableacid.
 11. Method according to claim 10, wherein said pH-marker is acompound of formula (II.1) in which R₃ is other than a hydrogen atom.12. Method according to claim 10, wherein said pH-marker is a compoundof formula (II.1) in which R₁ and R₂ are both other than a hydrogenatom.
 13. Method according to claim 11, wherein said pH-marker is acompound of formula (II.1) in which p represents
 0. 14. Method accordingto claim 13, wherein said pH-marker is a compound of formula (II.1) inwhich R₃ is selected from the group consisting of a (C₁-C₆)alkyl groupunsubstituted or substituted with one or more radicals selected from thegroup consisting of nitro, halogen, (C₁-C₆)alkoxy, (C₆-C₁₀)aryl and(C₃-C₈)cycloalkyl, and optionally bearing a group —P(O)(OR)₂ inposition
 1. 15. Method according to claim 13, wherein said pH-marker isa compound of formula (II.1) in which R₁ and R₂ are independentlyselected from the group consisting of a (C₁—C₆)alkyl group unsubstitutedor substituted with one or more radicals selected from the groupconsisting of (C₁-C₆)alkoxy, (C₅-C₆)cycloalkyl, halogen, (C₆-C₁₀)aryland nitro; a (C₆-C₁₀)aryl group unsubstituted or substituted with one ormore radicals selected from the group consisting of (C₁-C₆)alkyl,(C₁-C₆)alkoxy, halogen, nitro and (C₅-C₆)cycloalkyl; and a group—P(O)(OR)₂; and R is selected from the group consisting of a(C₁-C₆)alkyl group and a (C₆-C₁₀)aryl group.
 16. Method according toclaim 13, wherein said pH-marker is a compound of formula (II.1) inwhich R₃ represents a (C₁-C₆)alkyl group; and R₁ and R₂ areindependently selected from the group consisting of (C₁-C₆)alkyl andphenyl unsubstituted or substituted with one or more radicals selectedfrom the group consisting of (C₁-C₆)alkyl, (C₁-C₆)alkoxy, halogen, nitroand (C₅-C₆)cycloalkyl and a group —P(O)(OR)₂.
 17. Method according toclaim 16, wherein said pH-marker is a compound selected from the groupconsisting of 2-propylamino-2-diethoxyphosphorylpropane,N-[1-phenyl-1-diethoxy-phosphorylethyl]-N-propylamine andN-[1,1-bis(diethoxyphosphoryl)methyl]-N-tert-butylamine.
 18. Methodaccording to claim 10, wherein said pH-marker is compound of formula(II.1) in which p represents 1; R₃ represents (C₁-C₆)alkyl; A represents—CR₄R₅—; R₁, R₂, R₄ and R₅ are independently selected from the groupconsisting of a hydrogen atom and a (C₁-C₆)alkyl group; and R isselected from the group consisting of a (C₁-C₆)alkyl group and a(C₆-C₁₀)aryl group.
 19. Method according to claim 18, wherein saidpH-marker is N-[(1-methyl-2-diethoxyphosphoryl)ethyl]-N-n-butylamine.20. Method of pH measurement by NMR spectroscopy, comprises: measuringpH-marker in ³¹P NMR, wherein said pH-marker is compound of formula

 in which T′₁ and T′₂ are independently a (C₁-C₁₈)alkyl, (C₆-C₁₀)aryland —OR′ group; R′ is selected from the group consisting of a hydrogenatom, (C₁-C₁₈ )alkyl and (C₆-C₁₀)aryl; R′₁ is selected from the groupconsisting of a hydrogen atom; a deuterium atom; a halogen atom; a(C₁-C₁₈)alkyl group unsubstituted or substituted with one or moreradicals selected from the group consisting of (C₁-C₆)alkoxy,(C₃-C₁₁)cycloalkyl, halogen (C₆-C₁₀)aryl and nitro; a (C₆-C₁₈)aryl groupunsubstituted or substituted with one or more radicals selected from thegroup consisting of (C₁-C₆)alkyl, (C₁-C₆)alkoxy, halogen, nitro and(C₃-C₁₁)cycloalkyl; (C₁-C₁₈)alkoxy unsubstituted or substituted with oneor more radicals selected from the group consisting of (C₁-C₆)alkoxy,halogen, nitro, (C₃-C₁₁)cycloalkyl and (C₆-C₁₀)aryl; a nitro group; agroup —P(O)(OR′)₂; and a (C₃-C₁₁)cycloalkyl group unsubstituted orsubstituted with one or more radicals selected from the group consistingof (C₁-C₆)alkyl, (C₁-C₆)alkoxy, halogen and nitro; R′₂ and R′₃ togetherform a divalent radical;

in which the group —C(L₁)(L₂)— is directly linked to the carbon bearingR′₁ and in which L₁, L₂, L₃ and L₄ are, independently of each other,selected from the group consisting of a hydrogen atom; a deuterium atom;a (C₁-C₁₈)alkyl group unsubstituted or substituted with one or moreradicals selected from the group consisting of (C₁-C₆)alkoxy,(C₃-C₁₁)cycloalkyl, halogen, (C₆-C₁₀)aryl and nitro; a (C₆-C₁₀)arylgroup unsubstituted or substituted with one or more radicals selectedfrom the group consisting of (C₁-C₆)alkyl, (C₁-C₆)alkoxy, halogen, nitroand (C₃-C₁₁)cycloalkyl; (C₁-C₁₈)alkoxy unsubstituted or substituted withone or more radicals selected from the group consisting of(C₁-C₆)alkoxy, halogen, nitro, (C[3]₃—C[11]₁₁)cycloalkyl and(C₆-C₁₀)aryl; a nitro group; a group —P(O)(OR′)₂; or a group(C₃-C₁₁)cycloalkyl unsubstituted or substituted with one or moreradicals selected from the group consisting of (C₁-C₆)alkyl,(C₁-C₆)alkoxy, halogen and nitro; and L₅ and L₆ represent, independentlyof each others a hydrogen atom; a deuterium atom; a (C₁-C₁₈)alkyl groupunsubstituted or substituted with one or more radicals selected from thegroup consisting of (C₁-C₆)alkoxy, (C₃-C₁₁)cycloalkyl, halogen,(C₆-C₁₀)aryl and nitro; a (C₆-C₁₀)aryl group unsubstituted orsubstituted with one or more radicals selected from the group consistingof (C₁-C₆)alkyl, (C₁-C₆)alkoxy, halogen, nitro and (C₃-C₁₁)cycloalkyl;(C₁-C₁₈)alkoxy unsubstituted or substituted with one or more radicalsselected from the group consisting of (C₁-C₆)alkoxy, halogen, nitro,(C₃-C₁₁)cycloalkyl and (C₆-C₁₀)aryl; a nitro group; a group —P(O)(OR′)₂;and a (C₃-C₁₁)cycloalkyl group unsubstituted or substituted with one ormore radicals selected from the group consisting of (C₁-C₆)alkyl,(C₁-C₆)alkoxy, halogen and nitro; and a group —P(O)(OR′)₂; p′ represents0 or 1; A′ represents a divalent radical —CR′₄R′₅— in which R′₄ and R′₅have the meanings given above for R′₁ with the exclusion of —P(O)(OR′)₂;it being understood that the said compound does not contain more thattwo groups —P(O)(OR′)₂; or a salt thereof with a pharmaceuticallyacceptable acid.
 21. Method according to claim 20, in which R′ is otherthan a hydrogen atom.
 22. Method according to claim 20, wherein saidpH-marker is a compound formula (II.2) in which R′₁ is selected from thegroup consisting of a hydrogen atom, a (C₁-C₆) alkyl group, a(C₆-C₁₀)aryl group and a —P(O)(OR′)₂ and R′₂ and R′₃ together form aradical of formula:

in which L₅ and L₆ are as defined in claim
 20. 23. Method according toclaim 21, wherein said pH-marker is a compound of formula (II.2) inwhich L₅ and L₆ are independently selected from the group consisting offrom a hydrogen atom, a (C₁-C₆)alkyl group, a (C₆-C₁₀)aryl group and agroup —P(O)(OR′)₂, R′ being selected from the group consisting of a(C₁-C₆)alkyl group, a (C₆-C₁₀)aryl group and a hydrogen atom.
 24. Methodaccording to claim 21, wherein said pH-marker is a compound selectedfrom the group consisting of: 2-methyl-2-diethoxyphosphorylpyrrolidine;2,2-bis(diethoxyphosphoryl)pyrrolidine;2,2-bis(diisopropoxyphosphoryl)pyrrolidine;2,5-bis(diethoxyphosphoryl)-2,5-dimethyl-pyrrolidine;trans-2,5-bis(diethoxyphosphoryl)pyrrolidine; ethyl2-methylpyrrolidin-2-ylmethylphosphinate;2-phenyl-2-diethoxyphosphorylpyrrolidine; and2-methyl-2-diethoxyphosphoryl-5-phenylpyrrolidine.
 25. Method accordingto claim 10, wherein T₁ represents —OR and T₂ represents —R.
 26. Methodaccording to claim 11, wherein T₁ and T₂ represent —OR.
 27. Methodaccording to claim 20, wherein T′₁ represents —OR′ and T′₂ is selectedfrom the group consisting of (C₁-C₁₈)alkyl and (C₆-C₁₀)aryl.
 28. Methodaccording to claim 20, wherein T′₁ and T′₂ represent —OR′.